UN I TED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON , D

. C

. 20460



## A

UG 2 4 2 015 OFFICE OF CHEMICAL SAFETY A ND P O L LUT I O N PRE V ENTION



### MEMO RAN D U M

UB J ECT:

FR O M:



### PEE R REV IEW: 

### TO: Environmental Ri sk Assessment for a FlFRA ect i o n 3 Limited Seed In crease Re g i s tration of DvSnf7 Double Strande d RN A ( d s R NA) a nd Cry3Bb I 

### Ba c illus thuringiens is D e rived In sec ticidal Prote in as Ex pre sse d in MON 87411 Maize: E P A Fil e Sy mb o l 524

- A RI ; PC Codes: 006566 , 006580; Decision o. 487782; Submission os. 960 1 20 , 963404 , a nd 94746 1 ; DP Bar codes : 4 279 17 , 424 378, 4 26505, and 4 204 43 ; MRI Ds 4931 5 1-11 throu g h -19
. 493 15 1- 2 1 and

- 22, 49 5058 -0 2,

- 03, a nd

- 05, 495533-04 a nd

- 06: ubmitted by Monsanto Co mp a n y Shann o n B orges, Lead Bi o lo g ist

# ~~~ Mi cro bi a l Pes ti c ide s Branc h Bi o p es ti c id es a nd Polluti on Preven ti o n Di v i sion

_

### 'Jr' 

# ... t
Chri s Wozniak. Ph.D. , Bi o technolo gy S peci a l Ass i s ta nt 

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- 

# C**/{~ ~**  Biop cs ti c id es a nd P o lluti on Preventio n Di v i s i on 

# l'v'-" 1 L/ J e annine Kau sc h , Reg ul ato r y Action Lead e r Microbial Pest ic id es Bran c h Bi opcs ti c id es a nd Pollution Preven ti on Di v i s ion


### I. In t r o du c ti o n M o n s anto Co mp any ha s s ubmitted an app li ca tion fo r a FJFRA Sec tion 3 see d in c rease reg i s tration for l\'>' 0 n ew plant in co rporat e d protecta nt ( PIP ) ac tiv e in gre di e nts exp resse d in Eve nt MO 8 74 I 1 tran sge ni c co rn. Th e ne w ac ti ve in gred ients inclu de a d oub le s trand e d RNA (ds R A) tran sc ript ( D vS nf7 ) derived from western co rn rootwo rm 

### (Diahro t ica v ir g(fe ra virf?((era, W C R) a n d a lso a C ry3 Bb 1 in sec ticidal p ro te in deri ve d from 

### Bacillus thuring i ensis (Bt) s ub s p. 

### ku m a moto ens is. Th e D vS nJ7 dsRNA a nd Cry3 Bbl protein ex pre ss ed in M ON 8 74 11 confe r res i s tanc e to co rn rootworm 

### (Diab ro ti ca spp., C RW ). MON 87411 a l so exp resses th e 5- e n o lp y ru vy ls hikim a tc-3- pho sp ha te synthase (E P S P S) protein , w hich co nf e rs tol e ra nce to

### pho~ : ;J The D vS nJ 7 dsRNA res ult s from ex pres s i o n of an in ve rte d repeat seq u ence designed to m a tc h the se qu e n ce of W C R 

### Sn.f 7 ge n e . Express io n of th e se quenc e res ult s in th e formati o n of th e d s RNA tr a n sc ript co nt a inin g a 240 bp fr ag m e nt of the WCR 

### Sn.f 7 ( D vS nt7 ). S nt7 i s a vac u o lar

1so rtin g protein belon g in g to th e Endo so mal So rtin g Complex R eq uired fo r Transport (ESCRT)

- III co mpl ex. w hich i s involved in sor tin g of tran s membra n e prote ins 

### en route to lysoso m a l degradation through the e nd osomal- autophagic pathway in a numb e r of orga ni sms. Once co n s um e d , th e DvSnt7 d s RNA i s r ecognized b y the C R w·s RN A interfere nce ( R NA i) machinery w h ere in it i s cleaved into 2 1- 24 mer s mall inte rferin g RN As (s iR NAs). These s iR As b in d to a R NA induc ed s il e n cing compl ex ( RI SC) , w hi ch ul tim ate ly lead s to d ownre g ul a tion of the tar ge te d 

### D vSn.f 7 ge n e a nd m o rt a lity . The C ry3Bb 1 protein exp ressed by MO 87411 i s a d e lta-endotoxin from 

### Bacillus thuringiensis s ub s p. 

### kumamotoensis , w hi c h h as specific act i v it y aga in s t insect s within th e Ord e r Co leop te ra. E PA determi n ed that th e Cry3 Bb I in sec ti cidal pro te in exp ressed by MO N 874 11 i s b ioc h e mi ca ll y a nd fun ctionally eq ui va le nt to th e Cry3Bb l expressed in MON 88017 com (U.S
. EPA 2 015 )
. The activity of th e C ry3Bbl prote in e xpresse d in MON 88017 was a lso dete rmin e d to b e b ioc h emica ll y and functi o nall y equ iva l en t to Cry3Bb l expressed in MON 863 mai ze, and both we re d e termi n ed to hav e no unreaso n ab le adve r se effe cts on n o ntarget o rganis m s (U
. EPA 2 010a )
. An Ex perim e ntal Use Perm it (EU P ) was p r eviou s ly i ssued for M ON 87411 ( and a s imil ar PI P. MO N 874 10) a nd oth e r p rev io u s ly r eg i s te red PIP s in 20 1 3, ending in 20 1 5. Thi s E U P in vo lved approximate ly 1 3 , 000 ac res on w hi ch MO 8741 1 and MON 87410 were to be grow n , a nd the duration of th e EUP was two year s. Thi s EU P was exte nd ed in 20 14 until February 2016. a nd th e acreage in vo l v in g MON 87411 was ex panded to approx imate ly 19 , 000 acres (MON 87410 was n o lo n ger bein g tested in the EU P )
. The EU P was extended again in February 20 1 5 , but th e ac reage in vo l v ing MO 87411 was reduced to ap pro x im ate ly 9 , 500 acre s. The eco log ical ri sk assessment for thi s EUP reli e d primarily o n dat a demon s tratin g th e s p ec ifi city of the D vSnf7 d s RNA fo r th e WCR target pe s t as we ll as rati o nale de sc ribin g so me barri e r s to uptake of th e D vSn f 7 dsR NA in n o nt arget o rg anism s. In li g ht of the limited acreage involving MON 87411 a nd MON 87410 and th e limite d durati o n , E PA determin e d that the risk of adver se e ffect s to population s o f nontar ge t organisms was minimal. Du e to un ce rt a inti es of dsRN A pesticide ac ti v e in g re di e nt s id e ntifi ed in th e liter a ture and potentiall y assoc ia ted wi th human and nontarge t organ i s m risk s. E PA cons ulted w ith th e F IFRA Sc ientific Adv i so r y Panel (SA P ) fo r g uidance in unders tanding and address in g the se un ce rt ai nti es. The m ee tin g of the SAP w as he ld Janu a r y 2 8 , 2 014 , with minute s publish e d in May of tha t yea r 1 • The SA P cons ultatio n was h e l d ind epe n dentl y of an y r eg i s trat io n in vo lvin g a d s R A ba se d p est icide, including MON 87411. Mon santo Company has included th e ir res ponse to th e minutes of the SA P in th e in format ion s ubmitte d to s upp o rt thi s r eg i s tration ( MRID 49505801 ). Th e initi a l a pplication fo r MON 87411 s ubmitte d in 2 014 was for a seed in c rease r eg i str at io n wi th t he standard limit s of 20
. 000 acres per coun ty and up to a co mbin ed U.S
. tot a l of 250,000 acre s per Pl P acti ve in g red ie nt per r egis trant p e r year. In li ght or un ce rt ai ntie s di sc u ssed at the SAP m eetin g, EPA i ss u ed a 75 -day d e ficienc y le tter in Dece mb e r 2 014 re questin g additional data. In February 2015. Mo n santo req uested a c hange to the p roposed re g i strat io n by limitin g th e registrati o n to tw o years with 1 5,000 ac res of MON 8 74 11 grown in each ye ar. A respon se to the

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. \\ \\ ' \\ .cpn.;.?.ov**~t:ipo l\'**  sap**meeting,~** 20 I..\ i<muan 'O128 1 4111i1111 1 es.pdr 275-d a y le tte r 

### (M RlD 49553302) was also s ubmitted , w hich con tained so me additional data , as well as rationale s upportin g waivers for th e additional data r eq ue s ted. Th i s m emora ndum co ntain s the env ironmenta l risk assessment fo r O vSnl7 and Cry3 Bb I ex pre sse d in 

### MON 87411 for the prop osed seed in c rease r eg i s tra tion. D a ta s ubmitted for D v nf7 a nd data bridged from a previous registration of C r y3Bb1 (see discus s io n below) a re conside re d in the context of env i ronmental exposure r e levant to ex pre ss io n of th ese ac tive ingredients in MON 87411. Thi s in fo rmati o n and additional lin es of ev id e n ce a r e used to characterize ri sk fo r n o ntarg e t o rg anism s under th e limited r eg i s tration described above
. Pertin e nt information fro m the respon se to th e 75-day J e tte r and the res pons e to th e minutes of th e A P are in c lud e d in thi s ri s k asse ss m e nt. 

### II. 

### Ge n eral Approac h to Eco log ica l Ri s k Assessme n t for PIPs E PA · s c urre nt eco logical ri sk assessment approac h for PlP s was d eve lo p ed primaril y from ex perience with Br-de rived C r y and Y ip protein s . These prote in s a re ge n e rally understood to be s pecific to target pe s t s and r elated in s ect s within th e same taxonomic order , and w ith nearl y two decades of history indicating safe use. E PA co n s id e r s the current approach s uffi c ie nt for determining eco lo g ica l risk s of Br-derived protein PIPs , includin g the Cry3 Bb I protein expressed in Event MON 87411 co rn . Th i s approach i s described in severa l Biope s ticid c Registrati on Ac ti on Document s ( BRAD s) for C ry or Yip proteins (e
. g., see U.S
. E PA 20 10a , pa ges 65-69). To s umm a ri z e , the approach co nsists of a ti ered tes ting sc h e m e (Tiers I

- IV) focusing on detenn in at io n of hazard , and tes tin g i s based o n th e data req uirem e nt s for microbial pe s ticides published under 40 CF R 1 58
. 2 1 50 and 885 and 850 se ri es OCSPP Harmoni zed Gu idelin es . At Tier I, s tudie s a r e designed to be s implified a nd to est im ate of haza rd of several n ontarge t laxa under "wo rs t-case" ex posure co ndition s. A lack of adverse effec t s und e r the se co nditi o n s would pro v id e enough co nfid e n ce that there i s n o ri sk and n o further data wou ld be nee ded. Sc re eni n g (T ie r 1 ) maximum h aza rd dose te s t s a r e conducted at ex po su re concentrations several times hi g h e r (e
. g., > 1 

### OX when poss ibl e) than the hi g he s t concentrations ex pected to occ ur und e r reali s ti c field exposure sce nario s, \v ith mort a lit y as the tox icol og ica l endpoint. A thres h o ld of 50% mort a lit y i s u sed as a tri gge r for add ition al higher-tier testing. Wh e n screen in g s tudi es s u ggest potentially unacceptable risk additio n al studies a r e de s ig ned to as s ess hazard under mo r e reali s tic fi e ld expos ure co ndition s . Tiers II

- IV generally encompass d e finitiv e h aza rd le ve l d etermination s, c h ronic s tudies, and longer te rm g reen h o u se o r fi e ld testin g , and are implemented w h en unacceptable e ffects arc see n at th e Tier I scree nin g l evel. When sc ree ning te s ts indicate a n ee d for addition a l data, the OCS PP H a rm o ni zed Guideline s call fo r te s tin g a t increme nt a ll y lower do ses in o rd e r to es tablish a de finiti ve LD so and to quantif y the hazard. 

# A ri s k d e te rmination i s made by co mparin g the toxi co lo g ical e ndpoint to the estimated env ironm e ntal concentrat ion (EEC)
. In ad diti o n to the tox i c ity data , ad diti onal data a r e also co n side r ed re ga r d in g the e n v ironm e nt a l persis tence of Pl P s . as well as the potential for gene flow and development of weedi n ess . EPA req uire s laboratory data dem o n s tratin g the degradation of the PIP in so il s typical of ag ron om ic
3areas where the PI P crop is grow n. To asses s ge ne flow and p o tential fo r deve lo pment of weed in ess, E P A consider s several line s of ev id e nce re lated lo c haracteri s ti cs of the c rop plant , in cluding reproduction , presence of wild re l a tive s, and con tainmen t or ot her miti gatin g mea s ures. Becau se of uncertainties associated with th e potent ial for unexpected effects related to ex p osure to d sRNA , EPA raised que s tions to the SA P r ega rdin g the appl icability of the above approach lo dsRNA P IP s . The SA P found that thi s approach was not s ufficient to determin e r is k s to nontargct o rganisms, a nd s u ggested an a lternat ive framewo rk (see pages 61-64 of th e SA P minute s), w hi c h EPA i s c urrently eva lu ating. 

### Ill. No nt arget Organi s m E xpo s ure Asse ss m e nt Two se parate SAP re p ort s (Oc to b e r 2000 and A u g u s t 2002) reco mmend e d that non-targ e t te s tin g of PIP s ( in those cases. 

### Bt de ri ved protein s) s h o uld focus on in vertebrate species exposed to th e crop in which the PIP (s) will be exp ressed. E PA has prev iou s ly determ in ed, ba se d o n these recommendations, that non-target organisms with the g reatest exposure potential to PIP s in tr ansgenic co rn fields a rc beneficial insect s that feed on plant tissues (e.g. , pollen ), so te rrestri al invertebrates are the focus of n o ntarget o rgan is m testing a nd of PIP ri s k assessme nt s . Nonet he less, E P A has recogn ize d that expos ure to oth e r n o n tar ge t organ is m s can occur (e.g. , exposure to bird s b y co n s umption of pest in sec ts that have consume d the PIP ), and has req uired testing on representati ve species. The E P A ri sk assessment is centered on determ in ation of hazard at the field expos ure rates, w hi ch a r e typ i call y ba sed o n ex pressio n leve ls w ithin the plant for PIPs. For th e purp oses of th e non-tar ge t o rgani s m ri sk assess m e nt o fDv S nf7 a nd C ry3 Bbl in Eve nt MO N 8741 I com , es tim ated expos ures a r e based o n the hi g hest co ncentrati on of th ese active ingredients expressed in com ti ss ue (s) based on protein expression stud ies (M RI O s 493 I 5 104 a nd 49315105). Fre s h weight and dry weight m easureme nts of protein expression per gram of tissue are typ ically determin ed fo r PIP s, alt h ough estimates of expos ure are based o n dry we ight measurement s, as fre s h we ig ht mea suremen t s ca n be hi g hl y vari able, as confi rm ed by t he SA P in October 2000
. Mean leve l s of Cry3Bb I expressed in var io u s tissues of MON 87411 (M RID 49315105) ranged from 4 μg C r y3 Bb I l g ti ss u e dry weight (dw t). as expressed in g rain , to 340 μg Cry3 Bb Ilg tissue dwt. a s expressed in the whole plant sampled during the Y3-V4 development s tage. Range s or mean exp ress i on level s we re 170

- 270**~L g/g**  dwt in leaves, 75

- I 80 μ gig dwt in root, 120

- 3 40 μ gig dwt in w h o le plant , and we re me as ured at 2 1 μ gig dwt in s tove r, 4 μ gig dwt in grain , and 36 μg / g in p o ll e n . Se nesce nt r oo t , forage , a nd forage r oo t h ad co nc e ntration s of I 9 , 39, and 36 μg / g dwt of C ry3 Bb I , respectiv e l y. Leaf, roo t , and w h o le plant we r e sampl e d a t th e Y3-Y4, V6-V8. V I 0-Y I 3, and V 14-R I development stages , and expression ge nerally decrea sed over the sampling periods. The hi g he s t expression rep o rted was 460 μ gig dwt, w hi ch was the m aximum of th e range of expression leve ls measured fo r whole plant. The express io n levels o f Cry3 Bb I in MON 874 I I corn plant ti ss ues are comparable those of the Cry3 Bb I exp ressed in MON 863 corn , a nd a re lower than mean dry weight exp ress ion leve ls ofC ry3 Bbl in MON 88017 corn (U
. S
. E P A 2010a).

4Ex pr1.; ss ion of Dv S nt 7 dsRNA was similarl y determin e d in MON 8 74 11 ( MRlD 4 93 15104 )
. Ex press ion lev e ls we re ve r y low, and me an ex pression leve ls ran ged fro m 0.1 34 x I 0- 3 μ g O v nt 7 dsRN Ng ti ssue dwt , as d e termin e d in pollen , to 84.8 x I 

# o3 μ g DvSnf7 d s RN N g ti ss u e dwt in who le plant s ampled during the V 3 -V4 deve lopment s tage. Ra n ges of mean expres s ion lev e l s were 44.6 x 10

- 3

- 73.9 x I 0- 3 μ g/ g dwt in leave s, 6 .84 x 10-3

- 23 .9 x 10

- 3 μ gig dwt in root, 18. 5 x I 0- 3

- 84.8 x I 0- 3 μ gig dwt in whole plant, and we re mea s ured at 0 .677 x I 0- 3 

### μgig d w t in s tover , 0 .104 x 10

-3 

### μg i g dwt in g rain , and 0 .1 3 4 x I 0- 3 μ g/ g dwt in pollen. Senescent root , forage, a nd fora ge r oot had conce ntrati o n s of 1 .39 x ·10

-3
, 4.26 x I 

# o3
, a nd 2
. 37 x 10

- 3 μg /g dwt, resp ec tively. A s with C r y3Bbl, leaf , root , a nd w hol e plant were s ampl e d at the V 3- V4 , V6-V8 , VI 0-V 1 3, and V 14- Rl dev e lopme nt s tages, a nd expression ge n era ll y d ec reas e d ove r th e sa mplin g period s. T he hi g h es t ex p ression re p o rted was 2 1 3 x I 0- 3 μgig dwt, w hi c h was th e m ax imum of the range of exp r ess i o n m eas ure d fo r whole plant. 

### IV. No nt arget Effects Data A
. Overview of Data S ubmitted In th e abse n ce of PIP- s pecifi c ri s k as sess m e nt g u id a n ce, E PA ha s hi s to ricall y re quired applicant s fo r Pl P reg i s trati o n s to me e t th e 40 CFR P a rt 158 data req uirement s fo r microbi a l pestic id es. These requirements includ e tes tin g on birds , mamm a ls, nontarget in sec ts, h o n ey bee, plant s, a nd aquatic an im a l s p ec ies . The October 2000 SA P recommended th a t w hil e actual p la nt material i s the pre ferred te s t material , bacte ria-d er i ved ac tiv e in g r e di e nt i s a lso a va lid test s ub s ta nc e, pa rti c ularl y in sce nario s w h e re tes t animals do n ot n o rm a ll y consume co rn plant ti ss ue and w h e re large a mounts o f ac ti ve in g r e di e nt a re needed for maxim um hazard do se te s tin g. In s upp o rt of the MO 87411 r eg is tration s, L est s ub stances u sed in th e s u b mitted s tudi es in c lud e d bacteriapro duc e d purifi e d D vS nt7 d s RNA te st m a terial as well as plant ti ss u e (g rain) from MON 8741 I corn. Co mparativ e a naly ses s h owed the equivalence o r th e bacte ri a ll y-prod uc ed and purifi ed D vSnf7 d s RNA tes t material with th e active in g redi ent exp r essed in MO 87411 co m (U
. S. E PA 20 1 5). Additiona ll y, a lth o u g h it i s r ecom mend ed th at non-targe t testin g be co nducted a t a test d ose at lea s t I 

### OX the EEC whenever p oss ible , th e tes t dose mar g in ca n be less than I 

### OX where unce11a in ty in the syste m i s low o r w he r e hi g h concentrat ions o f tes t mate ri a l are n o t possible to achieve due to tes t organ i sm feeding habit s. BPPD m ay a l so a ll ow for tes tin g at lower dose s in cases whe re man y s pe c ies are tes ted o r tests arc very se n s iti ve, al th o ugh the concentratio n u sed mu s t excee d the EEC. Limit do s e te s tin g o n represe ntati ve organi s m s from seve ral ta xa was p e rformed w ith D vS nf7 in s upp o rt of the proposed MON 8 74 11 FIFRA Sec tion 3 see d increase reg i s trati o n. A s tud y to dete rmine so il degradation of DvSnf7 ha s a lso be e n subm iu ed. as well as a bioinformatics st udy and a G e ld s tud y of effec t s to n o ntar get a rthropods in ag ri c ultural e nvironment s . Rational e to add ress other data reque s ted , as we ll as a r es ponse to th e 20 14 SAP repo rt , were a l so s ubmitte d. The indi v idu al r es ult s for n o ntargc t organism and so il degradatio n tes tin g for Dv S nt7 d s R NA a rc sum m a ri zed i n Table 1. Th e s tudi es are d escribed in more detail below, a nd full r ev iews of eac h s tud y can be fo und in th e indi v idual Data Ev aluation R eco rd s . T o address data re quirement s for Cry3Bb I , M o nsanto Co mpany c ite d to s tudi es with C ry3 Bb I exp ressed b y o th e r co rn events ( MO 88017, MON 863) that ha ve bee n previously rev iewed by
5EPA. To a ll ow fo r bridging to the Cry3 Bbl data, in fo rm atio n mu s t be subm itted to E PA to s h ow that the protein exp ressed by MO N 87411 i s eq ui valent to the Cry3Bb I protein u sed in the cited s tudi es, the prote in i s n o t ex presse d at le ve ls hi g he r than previous l y assessed, a nd that no sy n e rg i s m exi s t s between th e C ry3Bb 1 and Dv S nf7 ex pre ss ed in MON 874 1 I. EP A d e te r mi n ed that th e C ry3Bb I ex pressed in MON 87411 co rn i s biochem i cally and fun c tiona ll y equ ivalent to th e Cry3 Bb1 exp r essed in MON 88017 corn , which i s si milarl y equivalent to th e Cry3 Bbl exp ressed in MON 863 co rn . The data s ubmitted l o s upport the registration of C r y3 Bbl in MON 880 1 7 and MON 863 are d i scussed in the updated BRA D fo r those eve nt s (U.S. E P A 2010a). Co ncerning equivale n ce of prote in exp r ession levels. Cry3Bbl protein express io n in MON 87411 i s comparab le to o r be low that of M O 88017 and MON 863 , as discussed above. Monsanto Co mp a n y h as additionally s ubmitted two stud ies on sy n e rg i sm b etwee n Cry3 Bbl an d D vS n f7, w hi ch i s s umm a ri zed in the table a nd discussed further below. 

### T a b le 

### 1. Summ ary o f da ta s u bm i tte d for Ov S n f 7 d s R NA a s ex pre sse d in MO N 874 1 1 co rn to co mp ly w i th d a t a r e quire m e nt s p u b li s h ed i n 40 CF R 

### & 1 5 8
. 21 5 0
. Da t a O P PT S Te s t Subs t a nce Res ul ts S umm a r y a n d Class ifi ca t io n MRID Req uir e m e n t G u id e li n e No. Avian dietary 885.4050 MON 87411 A 42-day dietary study s howed no adve r se 49315111 testing. corn grain effects on survival, broiler performance, or broi ler chicken, carcass y ield in broiler chickens fed a die t Gallus containing MON 87411 grain
. do m es/ icus C las s ifi cat io n : S u oo le me nt a l Av ian inha l ation 885.4100 N / A N o t r equired
. I nhalat ion i s not expected t o be a N I A tes tin g route by wh ich birds m ay be exposed l o DvSnt7 dsRNA. Wild mammal 885.4150 

### I A Studies with laboratory animals are sufficie nt to 49505804 testing determine risk to wild mammal s. Adverse 49505806 effec ts were not observed in a 28-day study with purified DvSnt7 dsRNA or in a 90-day stu dy with MON 87411 com grain inc luded in th e diet at 33%. Fres hw ater fi s h 885.4200 MON 87411 I n an 8-week study, consump ti on of M ON 49505805 test in g. corn gra in 874 11 cbrn grain at the exposure leve l tested c hannel catfish , (30% of th e diet) had no adverse effec ts on lc:talurus s urvival , weight gain, or diet conversion. punc:talus Cla ss ifi catio n : S uo n le mc nt a l Freshwater 885.4240 NI A Submitted rationale was adequate to conclude Co ntain ed invertebrate adverse effects are unlikely with the proposed within t esting seed increase registratio n . 4955330 2 C lass ifica t ion: Acce ot a blc Es tuarine and 885.4280 N IA Not required. Exposure in marine and/ or 

### N I A marine animal es tuarin e enviro nm ent s is not ex p ec ted to be te s ting s ignificant. Non-target pl ant 885.4300 

### N I A No t require d
. Ex pos ure to non targe t plant s i s 

### N I A te s ting expected to be minimal. Non-target insec t 885.4340 Microbially- No adve r se effects on s ur viva l, development t o 493 1 5 11 4 testing. lady produced adult s ta ge, ti me to emergence, or adult biomass beetle, DvSnt7 dsRNA were obse rved in C. macu/a/G fed I000 ng Coleomegilla Dv nf7 dsR A / g diet for 21 day s. muc:ulata C lass ification: S u pp le me nta l b u t uol!radea b le

6Data O PPT S T e s t S ub s t ance Res ults Su mm a r y a n d Class ifi cat io n MRID Requir e m e n t Guid e lin e No. Non-target in sec t 88 5.43 40 Mi crobiall y- No adverse effects on adu l t s ur vi va l in P

. 493 1 5 115 te s ting , parasitic produced foviolatus fed a 30% honey/ water so lution was p. Pediohus DvSnf7 d s R A containing I000 ng DvSnf7 dsR Alg diet in a .fiwiolatus 20-day s tudy. Cla ss ifi ca ti o n : S uppl e men ta l but up gra d eab le 1 on-target insect 885.4340 Microbially- o adverse effects on su rvival or rate of adult 49315117 t es ting, insidious produced e m ergence in 0. i11sidios11s nymph s fed 1000 n g n ower bug , Dv S nf7 d s RNA Dv S n f7 d s RNA /g die t in a I0-clay s tud y. Orius insidi o. ws C lass ifi ca t io n : S uppl e me ntal b ut u p!?.radca b le o n-target insect 885.4340 Microbially- No adverse effects on s urvival or r e production 49315118 (soi I arthropod) produced we r e obse rve d in F. c andida exposed to I000 n g t es tin g, DvSnf7 d s R NA Dv Snf7 ds RN A /g diet in a 28-day s tudy. sp ringtail. C la ss ifica ti o n : S u pp le ment a l b ut u p gra d ea bl e Folsomia c:andida No n-targe t insect 885.4340 M i cro biall y- No adverse effec ts on su rvi va l, deve l opme nt to 493 15 11 9 testing , carabid produced adult s ta ge. time to eme r gence. or adult bioma ss beetle, Poeci/us DvSnf7 dsR A were observed in carabid bee tle larvae fed I000 cha/cites ng DvSnf7 ds R NA/g diet in a 35-day s t udy. C la ss ifi ca ti o n : S uppl e me n ta l but uo !! r a d eab le Hone ybee 885.-1380 Microbially- o adverse effects were observed in survival or 49315112 tes ting, larvae produced development of larvae provided a s in g l e dose Apis m ell(fe ra DvSnf7 dsRNA eq uiva l e nt to 11 .3 ng DvSnf7 d s RNA per bee. Cl a ss ifica I io n : Acc c ota bl e I l oneybee 885
.-1 380 Microbiall y- No adverse efTects on su rvival were observed 49315113 testing, adults produced when honey bee adults fed o n a s ucrose so luti on Apis mell((era DvSnf7 dsRN/\ co nt ai nin g 1 000 n g DvS n f7/g diet for 14 day s. Clas s ifi c at io n : S u p pl e me n ta l b ut Un1> r adea b l e Ea rthworm 850.6 200 Microbial ly- o adverse efTects on s urvival o r percent cha nge 493 1 5116 toxicity, p rod uced in biomass in ea rthw o rm s exposed to 5000 ng Eisenia and r e i DvSnf7 dsRNA Dv S n l7 ds RN A / g a rtifi c ial soil in a 14-day contact study. C las s ification: S upo lc me nt al but uo2ra dca bl e So il fate and 885 .520 0 Mi crobially- B ased on Quantigene® 2.0 ana lysis of so il a nd 49315122 deg radati o n produced bioactivity mea su r ed in Southern co rn DvSnf7 dsRNA rootworm, DvSnf7 ds R NA de g rades rapid l y in Plus MO soil with 90% dissipa1ion time ranging 45

- 55 87411 plant hr. ti ssue C la ss ifi c ati o n : Acce ptable A.I. interaction N I A Microbial ly- The toxicity ofCry3Bb 1 prote in to L. 493 15121 (syne rgi sm). produced decemlineato does not change when exposed Co lorado potato DvSnf7 dsR A either to a lethal concen trat ion ofCry3Bbl bee tle , and Cry3 Bbl alone o r s imultan eo us ly ex posed to Cry3 Bb I Leptinotarsa and I000 ng D vSnf7 d s R NA/ g diet. decemlineata Cla ss ifi cati o n : Accco t ab le /\.I. interac t ion 

### N I A Microbiall y- No dilTerence in toxicity (LC so) was observed in 49505803 (sy nerg is m), produced SC R exposed to a leth a l conce ntration of Southern co rn DvSnf7 d sRNA Cry3Bb I co mbin ed with a sub lethal roo t worm. a nd Cry3 Bbl concentration (EC ,0 for growth inhi bit ion) of
7Data OPPT S Te s t S u bsta n ce Res ult s S u mmary a nd C lassificat ion MR I O R eq u irem e nt Guid e lin e No. Diahrmica DvSnf7 Ds RNA or vice versa. Testing with 11ndecimp1111ctata additiona l concentrations is necessary to howardi confirm the potential for syne rgism. C lassi ficat io n : S uoolementa l Field eva luation 850.2500 MON 87411 No evidence of adverse effects to non tar ge t 49553304 of non target corn plants arthropod population s or co mmuniti es were arthropod observed between MON 87411 corn plots and abundance genetically similar (except for transgenic traits) contro ls in field plots s t udied in the U.S.. Brazil , and Argentina. The data appear to be variable
. and the study is not likely to predict differences amon g treatm ents. C las s ifi ca tio n : S u pp le m en tal Non targe t N I A DvSnf7 No**exact ~**  2 1 nt matches were found when th e 49553306 organism Sequence 240 nt DvSnf7 sense st rand sequence was bioinformatic queried against transcriptorne sequence analy s is collections !Tom 23 nontarget organisms. Cla ss ific a ti on: S upplemen tal Non target N I A MON 87411 The document provides some information that is 49505802 o r

ga n ism usef ul to th e ri s k assessment, and information environmental was incorporated here in where pertin e nt. ri sk assessment C lassi ficati o n : S upplemental 

### B
. S tud y Summarie s for DvSnn d s R NA 1
. Bi r d s Avian Di e tar y Toxicity A 42-day f eed ing assessment using Cobb x Cobb 500 broilers 

### (Gallus domesticus) was conducted to co mp are the nutritional value of diets containing MON 87411 m aize grain and a ne ar isogenic conventional m a ize co ntrol NL6169 with s imil a r background ge n et ic s to MON 87411. Fi ve add itional di e t s containing mai ze gra in representatives of the population of commercial conventional maize varieties were included. N utrient analyses of eac h maize grain source were used to formu late diets having identical nutrient specific ation s, which were fed to the broilers 

### ad lib it um throu ghout the study. Treatment s were assigned to p e n s randomly wi thin five b locks of 14 pens eac h (seve n male and seve n female) with ten broiler s p er pen for a total of 70 pen s and 700 bird s. Broilers were weig h ed o n Day 0 a t the e nd of the s tud y just prior to proces s ing to assess ca r cass yie ld
. Pen feed int ake was determined for the duration of the study, and u sed to ca l culate feed to weight gain ratio. There were no biologically relevant differe nce s in broiler performance. carcass y ield , or mortalit y ( mortality ran ged from 0 -0 .8% ac ross all treatments) betwee n broilers fed diets co nt a inin g MON 8741 I maize gra in a nd those fed d iets con t aining near i soge ni c conve nti onal co nt ro l NL6 l 69 m a ize gra in. The study report does not s tate the percentage of gra in in th e di e t ; however. Mon santo Company's environmental risk assessment (M RID 49505802) states that th e percentage o f MON 87411 in the d iet was 57%. While th i s i s more than half of the di et, expression of D vSnf7 dsRNA in grain (0.104 x l 0- 3 u g/g ti ssue dwt) is much lower than in othe r 8plant ti ss ue s . Thi s le ve l of exposure i s n o t a maximum ha za rd d ose that i s typicall y required fo r Tier I nontar ge t o rgani sm s tudi es, but it is a lso uncl e ar w h e th er thi s amount i s re prese ntat ive of th e EEC, s in ce b ird s may be ex po s ed thro ug h routes other th a n cons umpti o n of co rn gra in (e.g., co n s umption of p es t in sec ts that h ave con s um e d leaf or o rh er ti ss u e)
. A lso, while th e o th e r va ri ab les mea s ured (e .g. , we ig ht s of vari o u s body parts) may indi cate the nutriti o n a l performan ce of th e g ra in , it i s unclear what re levance th ey ha ve to s urv ival o r rep ro duction of w il d bird s . While this s tud y does pro v id e so me in s ig ht into th e poten ti a l for adverse effects in bird s, p a rti cu la rl y fo r ex te nded e x po s ure period s (6 weeks), it s u se fulne ss in determinin g ri sk dep e nd s o n th e ac tual leve l of ex p osu r e to birds in th e fie ld. B eca u se of thi s limit a tion , th e s tud y was d e te rmin e d to be s upplemental. 

### A vi an lnhalation Toxicity Avian inhalati o n to x icity i s a co ndition a l data requirement of 40 CF R 158.2150. S inc e E P A d oes n ot a nticipate s ig nificant exposure of birds to D vS nt 7 dsRNA v ia inh a lat ion. th ese da t a are not re quired fo r re g i s tration of D vS nf7 d s R NA in M O 87411. 

### 2. Wild Mamma ls Two s tudies are avai labl e with w hi ch to assess the p o te nti a l toxicity of D vS n f7 in w ild m a mmal s, in cluding a 28 -d ay ac ut e oral tox ic ity s tud y with mice (MR ID s 49505804 and 49576 10 I ) and a 90-day feedin g s tud y with rats ( MRI O s 49505806 and 4957610 I ). The se s tudi es were rev iewe d by E PA a nd class i fied as acc e ptable (U.S E PA 20 1 5)
. In th e 28-day s tud y . mi ce we re dosed d a il y with DvSnf7 te s t material w ith p urity of I 00 % a.i
.. and no adve r se effec t s were observed that we re attributable to the tes t mate ri a l. This s tud y es t ablished a NOA EL of I 05 m g/k g/ day. I n th e 90-day s tudy , rats were fed a diet of 33% g rain , a nd n o treatm e nt-re lated effec t s were obse r ve d on morta lity, clinic a l s igns , fun c ti o nal obser vation a l battery. body wei g ht , body we ig ht ga in , food consump ti o n , food effic ie nc y, c lini cal path o logy, o rgan weig ht s, g ro ss pa thology
. o r microscopic pathology
. B ased on information descri bed by Mo n sa nt o Co mp a n y, th e OAEL was determined to be 1899 and 2303 m g/ kg tota l cage body we ight p e r day for m a les a nd fema les, respectiv e ly : however , th ese va lu es a ppear to d esc rib e th e NOA EL for M ON 87411 in the di e t , a nd not th e NOAEL for D vS nf7 d s RNA. 

### 3 . Fre s hw ater F is h a nd In ver tebrates 

### Fr es hwa t e r Fish Toxicity A die t a ry bioassay w ith c h an n e l catfi s h 

### (lctalu rus pwu : tatus) was cond u c te d to address th e T ie r I data r e quirem e nt for fre s h wa te r fish. This stud y was designed to eva lu ate the nutritional eq ui va le nc e of di e t co nta inin g maize g ra in from M ON 874 11 co mp a r ed to diet s co nt a inin g ma ize g rain from a n ear i s o ge ni c co n vent iona l contro l o r from eac h of fou r refe re nc e mai ze vari e ti es . The di e t s were formulated to con tai n 30% of th e appropriate mai ze gra in va ri ety, to co nt a in approximately 32 ± I % c rude prote in , a nd to be i so nitro ge no u s. G ro up s o f I 00 fi s h eac h ( five re plicates per g roup ) were f ed the appropriate diet dail y for eig ht weeks. After e ig ht w ee k s, th e r e we re no s tati s tically s ig nifi cant diff e re n ces amon g the treatm e nt g roups for foo d consump tion , weigh t g ain. o r diet co n ve r s io n. and grai n fro m MO 874 1 I com was considered

9to be n u triti onall y e qu iva le nt to co n ve ntio n a l m aize grai n s wh e n fed to c h a n ne l cat fis h . W hi le thi s information i s helpfu l, th i s s tud y ha s s imilar limitation s fo r determ ini n g to xic i ty in fres h wate r fi s h a s d esc rib e d fo r the broil e r c hi c ke n s tud y a b ove . In t his c ase, th e di et con s i s te d o f
· ju s t 30% o f g rain , in w hi c h r e la ti ve l y low leve ls o f D vS nt7 are ex pressed co mpare d to oth e r co m ti ss u es . T h e s tud y s how s th a t a d v er se e ff ec t s are n o t likel y b ase d on th e s tud y co ndition s, and t h i s inform a tio n is usefu l to the ri s k a ssess m e nt. H ow ev e r , th e c e rta inty of ri s k es timated from this s t u d y i s depe nd e nt on th e ac tu al expo s ure in th e fi e ld. Fo r thi s reason , th e s tud y was cla ss ifi e d as s upp lem e n t a l. Du e to un ce rt a inti es assoc ia te d w ith p o te nti al ex p os ure in aq ua ti c sys te m s to MO 8741 I p l ant materi a l a nd unkn ow n fa te of D vS nf7 in pl a nt ti ss u e, E P A r e qu es te d inform a ti o n o n de g ra d a ti o n o f D vS nJ7 in plant ti ss u e in it s 7 5

- day d e fi c ie ncy le tte r. 

### E PA a l s o s tated that i f D vS nf7 wa s s hown to p e rs i s t in p l an t t i ss ue , th en a Ti e r I ac u te fre s hwate r fi s h s tudy ( O CS PP g uid e lin e 88 5 .4 2 0 0) wou ld be requi re d to s ho w n o ac ute adverse effec t s . Th e catfi s h s t ud y above wa s s ubmitte d ind e p e nden t ly of thi s reque s t , a n d had n o t b ee n r ev i e w e d and c on s id e r e d at th e t i m e th e le tte r was w ritte n. Mo n sant o Co mp a n y r espo nded to th e r eq u es t w ith rati o n a le di s cus s in g th e ra pid d eg r ad ati o n o f 

### R A a n d D vS n t7 d s R A. E PA · s prev i o u s co nclu s io n s r egardin g e x p os ure to P l P s in a quatic en v iro nm e nt s, a nd lin es o f e vid e nce le adin g to a co n cl u s ion o f low like lih oo d of effe c t s. includ in g ph ys io log ical baJT i e rs to ex po s ure and res u lt s of bioi nforma tic s anal y s i s . Thi s in fo rmatio n i s ad e qu a te to addre ss p o te nti a l effect s to fre s hwater fi s h for th e proposed see d in c r ease r eg i s trati o n . 

### 4. Ma rin e a nd Es t u a rin e F i s h a nd In verte bra t es E PA h as prev ious l y dete r m in e d th at expo s ure to 

### Br de ri v e d P l P s in m arin e a nd e s tu arin e e n v ironm e nts i s n o t s ig nifi ca nt. At thi s tim e , the r e i s no inf o rmatio n to i n di cate that thi s ass umpti o n wo uld n o t a lso a ppl y to O vS n l7
. Th e r e fore , n o d ata w ere require d to addre ss tox ic it y of m a rin e o r es tuari n e fi s h a n d i n ve rt e brates . 

### S. No n ta r ge t P la nt s Whil e it i s uncl ear at thi s p o int wheth e r D vS nt7 may be prese nt in root ex u d a tes of MON 87411 co m . it i s l i ke ly th a t a n y D vS n t7 th a t e nte rs th e so il v ia thi s so urce o r b y re lease fro m plant ti ssues w ill rap idl y break down (see d i sc u ss io n of so il d eg ra d a ti o n be low)
. Th e refo re, s i g nifi cant ex p os ure to plant s i s n o t expec ted , a nd d a ta to a ddress n o nt arge t plant tox ic ity we re n o t r e quired
. 

### 6. No n ta r gc t In s ect s a nd O th e r In ve r te bra t es Non ta r ge t insec t s In a 35 -d ay di e tary to x ic ity s tud y, carabid bee tl es ( Poec ilus c ha/ ci t es ( Say]) we re e xpo s ed to D vS nt7 tes t material a t a n o m ina l con ce nt ra tio n of 1 ,000 n g/g of die t ( M RI D 49 3 151 1 9 ) . A wa te r assay co ntro l a n d a p o ta ss ium ar se n a te pos iti v e co ntro l w e re a lso u s e d. Expo s ure of t h e carabid b ee t le s to th e th ree di e t s wa s re pli cate d th ree time s with 2 0 i n s ec ts p e r d ie t fo r a tot a l o f 60 in sec ts per diet treatm e nt. A ll diet ary ex p os ur es we r e ini tia te d w ith th e fir s t in s tar lar v a e. S ur v iva l. deve lopme nt to ad ult s ta g e. d eve lo pm e ntal tim e ( d ays) to a dult e m e rge nc e . and a dult

10biomass were measured. Mean percent s urvival at test te rmination for the test , control , and positive control d iets were 93 %, 92%, and 65% r espec tiv e ly . None of the larvae fed the po s iti ve co ntrol diet developed to th e pupa s tage. The lowe r survival and inhibited development of in sec t s fed potass i um arsenate co nfi r med that th e test system was capable of de tecting tox ic effects through the di e tary exposure; however , in most other s tudi es te s t insects did not s ur v i ve th e positive control treatm e nt. The percent development to adult for larvae expose d sepa ratel y Lo test a nd assay control diet s averaged 70% and 75%. For the tes t and assay contro l d ie t treatment gro up s . the development time to adult emergenc e averaged 33 days. Adults fed te s t a nd control diet treatment s ave ra ge biomass was 31.9 m g a nd 32.3 mg, r espec ti ve ly . The s tud y report determined a NOEC of I , 000 n g/g of di e t; howev e r , the LCso is more typical ly determ ined for nontarget maximum ha zard do se s tudi es. The re s ults of this s tud y demonstrate that exposure to DvSnf7 _9 68 RNA at the maximum haza rd dose co nce ntrati o n of I000 n g/g diet had no adverse effect o n the s u rvival , deve lopm e nt and g r owth of the carab id be e tle , 

### Po ec ilus cha/cites. Wh il e the s tud y doe s provide u sefu l information about th e potential toxicity of D vS nf7 in this spec ies, the study was determined to be su ppl eme ntal , but upgradeable i f the following information i s provide d: 1) data showi n g typical mortality of 

### P
. cha/cites exposed to pota ss ium arsenate or explanation of the rel a ti ve ly hi g h s urvi val in thi s gro up , 2) cumulative raw data o n food consum ption (o r confirmation that larvae consumed all food between feedings), m o rtality, development ti m e, and raw data on adu lt bioma ss . These data are needed to confirm the re s u lts of the s tudy. Th ese data must be provided fo r use of this s tudy to s upport a full commercial registration of MO 8741 I. Toxicity of D vS nf7 was also determined in insidious flower bug 

### (Or ius insidiosus) nymphs in a study involving I 0 days of exposure (M RID 49315117). The in sec ts we r e allowed to feed 

### ad libitum on a d ie t conta i ning Ov S nt7 at I000 ng/g diet. A posit ive contro l di e t conta in ing l 00 μg of pota ss ium a r se nate per gram diet and a negative co ntro l diet were also includ ed. Eac h treatm e nt consisted of 40 indi v iduall y house d n y mphs , which w e re in i tially exposed as 5-day old nymph s . S u rv i va l a n d development we r e observed. Survival of th e test nymphs was 93% in both assay co ntrol and O vS nf7 treatme nt. D eve lopment to adult occ urred at 95% a nd 98% in th e ass a y con trol a nd Ov nf7 treatment. r espective ly. The average development time fo r tes t n y mph s to deve lop in to adults wa s I I. I I ± 0.15 da ys in th e assay co ntrol treatment and I 0.87 ± 0.13 day s in the D vS nf7 968 R A treatment. In co ntrast the 

### 0. insidiosus n ymphs fed th e potassium arsenate diet s h owe d

_ I 00% m or t a lity by day 1 

### O with I 3% of n ymp h s developing to adu lt s. The re s ult s of th i s study s h ow that continuous di e tary ex posure to DvSnf7 for 1 0 day s a t a concentration of I 000 ng/ g diet had no effec t on the s ur v iva l and development of 

### 0. insidious nymphs. The results of this s tud y are usefu l to the ri sk assess me nt , but the stud y is classified as s upplemental. but up g radeabl e becau se data are need e d to confirm the res u lts. Data requ ire d includ e : a) the le n gth of tim e that obse r vatio n s we re made b eyond the 10-day exposu re , b) raw data o n mo11 a lity, emergence. and de ve lopm e nt time , and c) food co n s umption dat a or clarification as to whethe r food was consumed comple te ly be twee n feedin gs. These data mu st be provided for use of this s tudy to sup p or t a full co mmerci a l reg i s trat ion of MON 8741 I. Larvae of the lady be e tle, ColeomegiL/a maculata (Coleoptera: Coc ine ll id ae), we re a lso tes ted in a 21-day study to determine th e potential toxi c it y of D vSn f7 to nontarget insects ( MRI D 493 I 51 14). Larvae were ex po sed to Dv S nf7 in a test diet a t a nominal concentration of 1000 n g/g of diet. A negat ive water contro l and a po s itive control of potassium arsenate we re also
11

### tested. Exposure to 

### C. maculata to the three diets was replicated three times with 20 individually hou se d insects per diet replicate for a total of 60 in sec t s per diet treatment. All dietary exposures were initiat ed wi th the first instar larvae , and the study was continued until all su r viving control and test insect s developed lo the adult stage
. Survival , development to adult stage, developm e nt time (days) to adult emergence, and adult biomass were observed. M ean percent s urvival at te st tennination for the test , negative control. and positive contro l diets we re 92 %, 90%. and 17 %. re s pectively. None of the larvae fed the po s itive control diet developed to the pupa stage. The percent development to adu lt fo r larvae exposed separately to test and negati ve co ntrol diet s ave r aged 92% and 90% , re specti ve ly , and development time to emerge nce for both treatment g roups was 15 da ys. Adult biomass for t est or control diet averaged l 0
. 2 mg for both treatments. The OEC was determined to be l 000 ng / g of diet. s ince that was the concentration tested. The re s ults of this s tudy demon s trate that exposure to DvSnt7 at the maximum hazard dose concentration of I 000 ng/g of diet had no adverse effect on the survival, dev e lopment , and g rowth of this s pecies. The se results a r e useful to the risk assessment , but the study was classified as supplemental, but upgradeable , because confirmatory information was not available. D a ta needed to upgrade th e s tud y include: I) raw data on cum ul ative mortality , development time , percent development to adult, and adult biomass for each replicate for each treatment, and 2) food consumption data and/or confirmation that food was consumed completely between feedings. The se data mu s t b e provided for use of this s tudy to support a full commercial re g i stration of MON 87411. A fo urth species , parasitic wasp 

### (Pediob iusfoveola tus) was also t es ted for toxi c it y to DvSnt7 in a 20-day study. 

### P .f oveolatus were exposed to O vSnt7 test material at 1000 ng/g in a 30% h oney/ water (v/ v) solutio n (M RID 49315115). An assay control treatment of 30% hone y/ water so lution and a positive control diet at 200 μ gig potas s ium arsenate in a 30% honey/ wate r solution were also include d. The treatments were rep licated four times with I 0 adult wasps per replic ate for a total of 40 wasps per treatment. All dietary exposu res were initiat ed with new ly emerged adults after approximately 24 hours of acclimation, and feeding 

### ad libitum was a llowed for th e duration of the s tud y . There was no mortality of 

### P
. fove olatus adu lt s that had fed the OvSnt7 treatment diet o r the assay contro l diet after 20 day s. In contrast, th e 

### P
. foveolatus adults fed the potassium arsenate treatme nt diet showed 100 % mortality at da y 12. The result s of this study demonstrate that continuou s dietary exposu r e to DvSnt7 for 20 days at a concentration of 1000 ng/g diet had no effect on the s urvival of the parasitic wasp, 

### P
. foveolatus
. The NOEC of the D vS nf7 for the paras iti c wa s p was l 000 n g/g diet; however , since the study was designed more like a limit te st, the LC so would be the typical endpoint exp res sed from this typ e of study. The s tudy is u sefu l to the risk assessme nt , but was determ in ed to be s upplemental , but upgrad eab le, s ince confirmatory information was mi ss in g . Additional information required includes: l) explanation for a discrepanc y in a sub-report describin g samples te s ted for test material stabi lity . 2) clarify the u se of spec ifi c grav ity in calculat in g the nominal do se, and 3) food cons umpti on data or clarification that th e wasps consumed all food between feedings. These data must be provided for u se of this study to support a full comme rcial re gis tra ti on of MON 87411. 

### Honey Bee Two s tudi es with honey be es were s ubmitted to s upport the registration of MON 87411, in c luding a s tud y with adults (MRI D 49315113) and anothe r wi th larvae (MRID 49315112). The
12study wi th adults evaluated the potential dietary effect s or DvSnt7 on s urvival in a 14-day co ntinuous feeding study. Newly emerged**adults( ~**  2 da y old) were exposed to D vS nt7 at a concentrat ion of I 000 ng/ g, an assay control (no treatment), and a po s itive control (potassium arsenate at 20 μg / ml), each of which was present in a 50% s ucrose/ purified water (w / v) so luti o n. Each treatment g roup includ ed 80 adult bees in four replicates of 20 bees per r epl ica te (one replicate per cage). Each cage was provided with approximatel y 10 ml of the appropriate treatment diet so lution and bee s were allowe d to feed 

### ad libitum. Be es from each treatment gro up were observed daily for mortality , abno rm a l behavior, and appearance. No adver se effects on s ur v i va l, behavior and appearance were observed, and a NOEC for thi s s tud y wa s determined to be I 000 ng of DvSnf7 test material/g diet according to th e s tudy report. This study was class ifi e d as s upplemental but upgradeabl e . becau se food consumption data and/or clarification as to whether the diet was completely co n sumed by the bee s between feed in gs was n ot provided. The se deficiencies do not affect th e usefuln ess of the stud y for this proposed registration , but thes e data must b e provided for use of thi s s tudy to support a full commercial re g i s tration of 

### MO 87411. ln a 17-day dietary toxicity study, hone y bee larvae were exposed to a si n g le dose of DvSnt7 tes t materia l prepared a t a concentration of 1000 ng/g and delivered in a I 0 μI aliquot of 30% (w/ v) s ucro se/ purified water to each larval cell for a total ma ss of 11.3 ng DvSnf7/ ce ll. Add ition al treatment group s included an assay contro l (30% sucrose so lution ) and a positiv e control (30% so lution co ntainin g potassium a rsenate at 2,000 μg / ml). Larva (2 to 3 days old) in all gro up s were ex posed to a si n g le 10**~ti** dose of th e app ropriate treatment at s tudy initiati o n and observed over the course of larval/pupal development and adult e mergence. Each treatment group consisted of 80 h o ne y bee larvae in four re plicates of20 hone y bee l a r

vae per r ep licate. There was I 00% survival in hon ey bee larvae treated wi th e ith e r DvSnf7 o r th e assay con trol. Emerge n ce of adult hone y bee s from the test larvae in both the DvSnl7 and the assay control was initiated on the same day (Day 14) , approximately 50% on Day 1 5, and reached I 00 % on Da y 1 7 in both treatment s. There was n o s urvi val in the po s itive control confirmi n g the valid ity of the expos ure system. Observa ti o n s of newl y e me rged adult bees indica te d no adver se behaviora l o r morphological effects. Thus , the NOEL for thi s stud y was determined to be 11.3 ng of Dv Snf7 per larva, based on the study repo rt. The LD so i s more typ ica ll y r epo rt ed in nont a rge t studies de s ig ned to test a maximum hazard dose. This study i s consistent with s imilar s tudies accepted by E PA in the pa s t. and i s c la ss ified as acceptable. 

### S oil Inv e rtebrates In a 14-day contact s tud y. ten adult earthworms(£. 

### andrei) were exposed to a s in g le app li cation of D vS nf7 incorporated into an artifi cial so il s ubstrate at a rate equivalent to 5000 ng R A per g soil dry weight. No food was provided during the duration of the te s t. The test trea tment was compa red to a control treatment of purified water added to the soil. M o rtality wa s assessed over the 14-day testin g period , and the c han ge in fresh weight of the worms was assessed for s urvivors at 14 da ys after treatment. Th e re was no observed mortality a fter 14 da ys. The mean percentage change in bioma ss was -9.4% and -8.4 % in th e control g roups and tre atment gro up, re s pectively. Thi s s tudy wa s c lass ified a s s upplemental but upgradeable. This g uid e lin e (OECD 207) i s intended to tes t toxicity to earth wo rm s via contact expos ure. It i s unclear whether thi s route of exposure is appropriate for toxicit y te s ting with double s trand ed R A. and a dietary
13

### s tudy may ha ve been more approp riate fo r a test with thi s o rgan is m. A lso, it is unclear whether the DvSnt7 te s t m ate ri a l wo uld have degraded d urin g the 14 da y ex p osu re period , and there i s no ass urance that the co nce ntrati o n in the so il was co n s is tent throu g hout the s tudy. Th e gu id eline a ls o states specifically that multiple concentration s mu s t be tested to establish a NOEC. The s tudy could b e upg r a d ed with ju s tification that the ex po s ure rout e is re leva nt , and that the DvSnf7 was stab le throu g hou t the durati o n of the s tud y. These data mu st be provided for u se o r this study to s upp o rt a ful l com m erc ial registration of MON 87411. Tox icity to soi l in verteb rates was also t es ted wit h a 28 -day dietary tox ic ity s tudy of D vS n t7 to sp ringtails 

### (Folsom ia ca ndida). D vS nt7 t es t mate rial was incorporated into an inactivated yeast medium diet with a conce ntrati o n of l 000 g DvSnf7 per gram of diet. T hi s t est diet was compare d to an untr eated co ntrol diet of inac ti vated yeas t , a blank ( n o food) diet , a nd a to x ic 

### r efe re nc e diet ( in act ivated yeast treated with th e in sec t g rowth re g ulat o r, teflubenzuron). Sprin gtail s were a ll owed to fee d 

### ad libitum. At 28 days, there were no s ig nificant e ffect s on the s urvival o r reproductive capacity of the D vSnt7 treatment g roup co mp ared to co ntrols. Mortality in the untreated control. b lank diet, and r eference diet was 0%, 0%, and 93%, respect ive ly . The mean number of pro ge ny in the treatment g r oups was 169 fo r the untreated contro l diet , 0 for th e blank diet , 167 for th e D vS nt7 diet , and 0.3 for the reference diet. Th e NOEC , based o n mortality, was 1000 n g DvSnt7 per gram of diet according to th e stud y report. H owever, s in ce the stud y i s desig ned more like a limit o r m ax imum hazard dose st ud y, more typical endpoints determined from th ese data are th e 

### LC so a nd 

### ECso. T hi s study is u sef ul to thi s ri s k assessment , but was determin ed to be s upplemental , but up gradeabl e, since confirmatory data were not provided. Data th at a r e r e quire d to up g rad e th e s tud y include: 1) data o n food consu mp t ion or co nfim1ation that a ll food was co n s um ed between feed in gs, and 2) raw data o n m o rtality and reproduct ion. These data must be provided for u se of this study to support a full co mmercia l regi s tration of MON 87411. 

### Field Evaluation ofArth ropod Abundance A field s tud y ( MR!D 49553304) was conducted to determine w he ther M ON 87411 maize produces adverse effec t s on nontarget in sec t p o pulations across three geographic location s : U nited States, Argentina , a nd Brazil. Four to s ix s ites were cho se n within eac h geographic locat ion, and w ithin each site MON 87411 and a co nve nti o nal co ntrol (w ith sim ilar ge ne ti c backgrou nd excep t for th e insecticidal and he rbicide tolerant traits) were te s ted fo r difference s in n o ntarget arthropod abundance and nontarge t pest damage. Each site included plots w ith MO 874 l l. the co n ven ti onal control , and four conventional reference m aize va rietie s (w hich varied across si tes). At eac h s ite, nontarget arthr opod ab und ance was determined using bo th s ticky trap s a nd v is ual counts , a nd counts were conducted multiple times over th e g r ow in g season. Pes t damage was also assessed u s in g methods specific to eac h pest , and damage was assessed severa l times over the g row in g seaso n. Out of 121 individual s ite compari so n s, on ly fiv e s ignificant differences we re d etected between MON 87411 a nd the conve nti ona l co ntr o l in nontar ge t art hropo d abundance. Fo r pest damage , 56 comparisons were made , a nd s ig nifi can t diff e re nces betwee n MON 874 11 and the co nventional control we r e detected in three . These diffe rences were determined no t to b e bio logically significant.



### 14

### This sludy provides some informalion on polential effects lo arthropods in a more realist ic lield setting; h oweve r, the study design presents uncertainties in the data, wh ich limit s conclusions that can be drawn. It is unclear that the study, as designed. wou ld detect differences and provide strong indication that adverse effects do not occur in the field. Limitat ion s include: I) Abundance data are represe nted as counts of arthropods a t th e taxonomic le ve l of order o r fami ly, and some of the se taxa include man y species th at likely vary in th e ir ecolog ical funct ions and intra- co mmunity relationships. Natural variation in spec ies abundance may a l so ex i s t, and differences in sensitiv ity to the treatment may also vary among species. Pooling them removes the abil ity to account for s uch variatio n in the data, and increases the difficulty of detec t ing treatment effects. 2) Plot s i zes were r e lat ively s mall , and it is unclear whethe r the plots we r e imm ed iatel y adjacent or separated w ithin the si t e . This is impo r tant for understandin g the inde p endence of the data between treatments, s ince more mobile arthr opods could move between plots with different treatments. It 

### is also unclear whether the plots were in the vici nity of other corn that might serve as sources of the insects observed in the study. This is important to know, because if there were adverse effects that wou ld otherwise reduce cou nts within MON 87411 plots, they may be replaced by ot h er migrating in sects that co uld end up being counted in th e cens us . 3) Mean counts that were determined to be s igni fi cantly different between MON 87411 and the controls are compared to the range of means determined within the reference plots. In most cases it appears that the means for MO 8741 I and the contro l plots are consiste nt wi th the r eference ranges. However, in some cases both means for MON 87411 and the conventional control are outside the reference range and in others the range is very wide. This may br ing into question the re lian ce on the reference mean ran ges as a check against the t est for stat ist ica l s ignificanc e, and may be an indi cator of genera l va ri at ion in the data that makes it di fticult to d etect differences among treatments. 4) Pest damage could indicate that certain nontarget species are not affected by the insecticidal substances in MON 87411, especially since the insects are known to have fed o n the plants. Howe ve r , the values presented are low in some cases, a nd it is unclear whether enough data were collected to detect differences amon g treatments. The study r epo rt indicates that pesticides were app li ed in the s tud y. and it is un c lea r how thi s may have affected the results. Clarification of some of the above points would be helpful in the evaluation of this study
. The data do provide some indication that adverse effects to nontarget arthropods may not be highly likely. How eve r , the results are relativ e ly weak co mpared Lo a h ypo the sis driven fi e ld study conduc ted at an appropriate scale, and a field s tud y w ith a more robust design wou ld be needed to demonstrate unequivocally a lack of effects in the field. For this reason, this study was detem1 in ed to be supplemental. 7. D eg ra d a ti o n In So il 

### A laboratory study was performed to determ in e the aerobic degradation rate o r the DvSnt7 RN A in soil (M RID 49315122). 

### Microbially

- produced DvSnt7 test material or a control s ub stance was incorporated into soi ls collected from three different states in the U.S
.. Illinois ( IL ), Missouri
15

### (MO) and North D a kota (N D ). The DvSnf7 treatm en t a lso included MON 87411 le af materi a l. The USDA so il classifications were Si lt Loam ( IL) , Loam y Sand (MO) and C lay Loam (ND)
. The so il s differed in th e ir physicochemical characteristics (s ilt , san d and clay composition , pH , organi c matter , etc
. ) based o n a pre-study an a lys i s . Soi l sampl es were extracted and anal yze d for DvSnf7 co nc entration s using Qu anti Ge ne® 2.0 kits obtained from Affi metrix , Inc . [n addition , the decline in th e insecticida l activity was quantified by measuring the mortality of in sec t s exposed to ex tract s of th e soi l sampl es , u si ng a di et-incorp orat ion insect bioassay w ith the so uth e rn co m rootworrn. The es tim ate d DT s o and DT90 valu es for degradation of Dv S nf7

_ 968 in soi l based on QuantiGene molecular ana lys i s we re the fo llowin g : DT s o of 19 hr , 2 8 hr , a nd 1 5 hr and DT9o of23 hr , 35 hr , and 22 hr for IL , MO , an d NO, res pectively
. The dec l ine in bi ological activi ty of D vSnf7 in so il , meas ured aga in st s tandard co nc e ntration curves in th e SCR b ioassay , produced estim ated DT so and OT9o va lu es of 18 hr , 29 hr , 

### 14 hr and 23 hr , 

### 34 hr , and 2 1 hr for IL, MO , and ND res p ectively. T he bioassay d ata were also examin ed using a standa rd 3- paramete r l og is ti c eq u ati on to c haracteri z e th e decline in insect mortality against in c ub at io n time, w ithout conversion to so il concentrations. This approac h produced D vS nf7 dissipation curves comparab l e to those ob ta in ed from th e standard conce ntration analyses with QuantiGe ne and insect bioassay
. ln thi s evaluati o n , the est im ate d tim e to 50% dissipation of in sect m o rta lity was 23 hr , 33 hr , an d 22 hr , and the 90% dissipation time was 55 hr , 45 hr , and 45 hr for I L , MO , and N D , respective l y. Res ult s from th e so il degradation s tud y indicate that D vS nf7

_ 968 degraded within approximate l y 2 days afte r application to so il , regardless of texture , pH , clay conte nt and other so il diff e re nc es, as measured b y molecular an alysis (QuantiGe ne) and by in sec ticidal activity (bi oassay). Therefore, th e s tud y s upp orts the conclusion that the ds R NA tran sc ript deri ved from D vSnt7 suppress ion casse tte of MON 87411 is unl ikely to persist o r acc umulate in so i l und e r co nditi o n s simi lar to those s imulated in the experiment at l eve ls above analytic detection. There was a low leve l of bio logical activity beyond the time that the dsRNA degraded below the quant i fication limits of the QuantiGene analysis . It i s unknown whether th e residual activity was a result of th e D vSnf7 that was add ed to the so il , or the release of D vSnf7 from degrading plant tissue. H e nc e, there is so me uncertainty regarding the persistence of Ov S nf7 RNA from M ON 87411 at low , b ut biologically active levels. Further investigation woul d be need e d to confirm that a low leve l of biological activity does not continue b eyo nd t wo da ys ; howeve r, g ive n the rapid de g radati o n , a ny residual activ ity is not expected to continu e for very lon g. Thi s st ud y was dete rmined to be acceptab le . 8 . Synergi s m As d isc u ssed a bov e , the potential for syner gist ic effects must be evaluate d to allow brid g in g to data previously s ubmitte d o n Cry3Bb 1. Two s tudi es we re s ubm itted to address th i s requirement. 

### Syne r gism in SCR One stu d y was su bmitted that tested th e p o t e nti a l for syne rg i s m in the se n s iti ve insect spec ies, sou th e rn corn rootworm (M RID 49505803). The stud y was conducted as a 12

- day bio assay. Th e potential for interaction between D vS nf7 and C r y3 Bbl was evalu ated by comparing th e LC so valu es of a le th al leve l of C r y3 Bb 1 in th e presence of Dv S nf7 at a fix ed s ub-lethal concentration
16a nd a le lhal leve l o f D vS nl7 in lh e pre s ence o r C r y3 8b l at a tix e d s ub-l e thal co n ce ntration. Th e fixed s ub-l e thal conc e ntrati o n fo r Dv S n f7 and th e C r y3 Bb 1 p ro te in u s ed in th e two binar y mi x tures was e qui val e nt to a n EC10 valu e fo r g r ow th inhibiti o n de te 1mined in m e th od developmen l b ioassays . Cry3 Bb I a n d Ov nl7 were a lso lested at let h al leve ls a lo n e ( n o t as part of a b in a ry mi x ture)
. Ass a y a nd b u ffe r co nt ro ls we re in cl u ded, and m o rt a lity was low (7%

- 11 %)
. Th e SC R co nce nt ra ti o n res p o n ses fo r th e C r y3 Bbl pro te in a lo n e a nd th e C ry3 Bbl prote in in th e prese n ce o f a fi xe d s ub-l e th a l c o n ce ntra tion of O vS n f7 we re n earl y id e nti ca l and thi s i s re fl e cte d by co mparabl e LC so valu es for th e C ry3 Bb I protei n a lo n e a nd th e bin a ry mixture ( L Cso= 46.28 μ g/ml di e t for C r y3 Bbl al o n e a n d 40.65 μg/ ml fo r C ry3 Bbl co m b in e d w ith D vS n t7 a t a fixe d s u b -l e th a l co n ce ntra ti o n ). T h e SC R co n centra ti o n r espo n ses fo r th e D vS n f7 a lone a nd DvS n f7 co mbin ed w i th Cry3Bb 1 a t a fi xed s u ble tha l co n ce ntra ti o n we re a l so ve ry co mp a rab le (LCso= 6. 35 n g/ml di e t fo r O vS n f7 a lo n e and 8
. 1 6 n g/ml diet fo r D vS n f7 co mbin ed w ith th e s uble th a l conc e ntra tion of C r y3 Bb I )
. Th e s tud y auth o r s co n c luded th a t th ese data s how a lack or sy n e rg i s m b e twee n D vS nf7 and C r yJ Bb 

### l. E P A h a d prev io u s ly ra i sed co ncern s a b o ut th e use of th e fixed subl eth a l co n ce ntrat io n th at was s t a te d to be th e limi t a t w hi c h ove rt tox i c s ig ns wo ul d be obse rved. Mo n sant o Co mp a n y ju st ified thi s app roac h b y citin g to s tudi es in which fix e d s ubl e th a l co nce ntr a tion s h ave bee n u se d s ucc ess full y to a sse s s for a nd c h a racteriz e sy n e rg i s tic interac ti o n , includin g T a b as hnik ( 19 92) a nd c it a ti o n s within , a s we ll as B e ld e n and Lydy (20 0 6). Th ese pap e r s d o d esc rib e e x p e rim e nt s in w hi c h fi xed s u b le th a l conce ntra ti o n s h ave bee n u se d , eve n in very s m a ll a m o unt s (e
. g .. Macintosh ct a l. [ 1 990), c ited i n Tabas h n i k [ 

### 1992]). H owever. these pape r s a lso desc rib e ad di t io n a l tes tin g at multi p le co n ce ntrati o n s. de te rmin a ti o n or sy n e r g i s m based o n o b se r va ti o n s o f s ubl e th a l e ff ec t s, an d/o r a dju s tm e nts in th e ta rge t mo rt a lit y in th e co mp o n e nt prese nt in hi g h e r co nc e ntra ti o n ( e . g . , re duc e d to 10- 2 0 % in M ac i nt os h e t al. (1 990 ])
. Th es e a pproac h es bette r c h arac te ri ze th e n a ture o f th e p o te ntial interac ti o n , a nd e n s ure th a t a n e ff ec t wo ul d b e o b se rve d i 

## r it we re to occ ur. Whil e th e ap p roac h u se d in thi s s tud y is n o t un l ik e some of th ese o th e r s tudi es, a nd m ay be va lid , th e re i s s till un certain ty as to w h e th e r a synerg i stic effec t o n m o rt a lity co ul d be o b served a t th e s ubleth a l l eve ls o f D vS n f7 o r C r y3 Bb I tes ted
. A dditi o n a l test in g u s in g s ubl e th a l e ndp o int s (e
. g., growth inhibiti o n ) or tes tin g bo th Cry3 Bbl a nd D vS n f7 a t multipl e c onc e ntrati o n s or medi a n l e th a l lev el s w o uld r e du ce thi s unc e rt a inty . T hi s s tu dy i s c lass ifi e d as s uppl e m e nt a l , a nd i s s u llic ic nt to s upp o rt th e p rop os ed see d in c rease reg i s trati o n. Ho w e ve r. a n ad d i tio n a l s tu dy bette r des ig n ed to d e tec t sy ne rgism b etween Cry3 B b 1 a nd DvS n f7 i s re quired to suppo rt a f ull co mm e rc i a l reg i s tr a ti o n fo r ION 874 11. 

### Syn e r gis m in Co lo rado P o t

a to Bee tl e A n a dditi o n a l c onfirm a to ry s tud y w a s s ubmitte d to ev alu a te th e po te nti a l fo r in te ra c ti o n b e twee n th e O vS nf 7 a nd C ry3 B b l pro tei n u s in g Co lo rado po ta to bee tl e (C BP , 

### Leplino ta r sa dece m linea t a) in a 1 2

- day co ntin uo u s feed in g b ioassay ( MR IO 49315 1 2 1). Th e Colo rad o p ota to bee tl e i s n o t s u sce p tibl e to D vS n f7, acco r d in g to B ac hm a n , e t a l. (20 1 3). T he p o te nti a l fo r inte rac ti o n w as eva luate d b y co mp a rin g th e LC so va lu es an d th e 95% c onfi de n ce inte rv al s o f th e Cry3 Bb I prote in in th e prese n ce a nd a b se nce o f D vS nf7 at a fi xe d c onc e ntr a ti o n o f 1000 n g / m L d ie t. No min a l tes t co n ce ntr a ti o n s for th e C ry3 Bbl pro te in we re b a se d o n th e res ult s o f prev io u s b i oassays p e r fo rm e d a t th e tes tin g fac ility. Mean m o rt a lity in a ll assay a nd b u ffe r co ntro ls was I 0.42% at assay te rm in a ti on. Co lo ra d o po t a to bee tl e de m o n s tra ted n ea rl y id e n t ica l
17

### conce ntration- effect relat ion ships to the Cry3Bb l protein alone and in the presence of fixed concentration of DvSnf7. The study is acceptable as a confirmatory study of no interaction in a nontarget spec ie s , but does not definitively test interaction because it is unclear whether CP B has any sensiti vity to OvSnf7. 9 . Nontarget Organism Bio i nformati c Analysis 

### At the request of E PA , Monsanto Company submitted a bioinformatic evaluat ion of the DvSnt7 se quence , wherein the 240 nt DvSnf7 sequence was compared to DNA transcriptome databases for 23 nontarget organisms (MRID 49553306 )
. The evaluati on was conducted u s in g STELLAR sof tware version 1.3 (Kehr et al. 2011) to identify exact 2 1 nucleotide matches between the DvSnt7 query and sequences contained in collections fo r the nontarget organisms , which were: 

### An as platyrhynchos (mallard), 

### Apis mellifera (honey bee) , 

### Bombus terrestris (bumb le bee) , 

### Bos taurus (catt le), 

### Caenorhabditis elegans ( nematode), 

### Canisfamiliaris (domesti c dog) , 

### Columba livia (roc k pigeon) , 

### Danaus plexippus (monarch butterfly) , 

### Dania rerio (zebra fish), 

### Daphnia pulex (water flea), 

### Drosophila melanogaster (commo n fru it fly) , 

### Equus caballus (horse) , 

### Gallus gallus (red junglefowl/chicken) , 

### Hyalella azteca (scud) , 

### Locu s t migratoria (migratory locust) , 

### Megachile rotundata (alfa lfa leafcutter bee) , 

### Mus musculus (ho u se mouse) , 

### Nasonia vitripennis (jewe l wasp), 

### Oryzias latipes (medaka), 

### Rattus norvegicus (Norway rat), 

### Solenipsis invicta (red fire ant) , 

### Sus scro.fa (pig), and 

### Tetranychus urticae (sp id er mite). No exact matches of 21 nt o r greater were found with any of the sequences searched
. This bioinformatic a nal ysis provides some add iti onal information toward another li ne of evidence of no expected adverse effects in nontarget organisms. Of these , the honey bee and chicken have a l so been tested again st exposure to D vSnf7 , and no adverse effects we re identifi ed in those st udi es. The report for the bioinformatic evalu ati on does not provide details about how these spec ies vvere chosen, nor does it include searches of other co leop terans that wo uld be more closely related to the target spec ies . Bioinformatics analyses are not considered to be predictive of effects. EPA is currently evaluating how bioinformatics may be used in nontarget ri sk assessme nt s, and at this time this analysis is considered to be supp lemental information. V. Risk Characterization and Descri ption A. Risk of Nontarget Organisms to Cry3Bbl Expressed i n MON 87411 Corn 

### Monsanto Company has cited to studies previously submitted to support the registration of MON 88017 corn. MON 88017 expresses a Cry3Bbl protein that is equ ivalent to the Cry3Bbl expressed in MON 87411. As discussed above, EPA previously determined that the Cry3Bbl exp ressed in MON 88017 is equiva lent to the Cry3Bb l 

### expressed in MON 863 , and much of the data submitted to suppor t MON 863 was bridged to support the registration of MON 88017. Expressio n of the C ry3 Bb I protein in MON 87411 does not exceed that of either MON 863 or MON 88017. The synergism study conducted with a sensitive test species does not s how definitively that a synergistic relationship does not exist between the Cry3Bb 1 and DvSnf7 in MON 87411, and additional data on synergism i s required to support a full comme rcial registration for MON 87411. H owever, the data available plus preliminary data provided from an additiona l s tud y o n growth inhibition in com rootworm (described in MRID 49505802) indicate
18

### that sy ne rg is m is unlike ly . T h e refo re, brid g in g to data to s upport registrations o r th ese other eve nt s exp ress in g Cry3Bb I is ap propri at e for thi s reg is trati o n . Acceptab le data ci ted s howed that ad ve rse effec ts a re not expected to occur for birds, wild mammals , fres hwater fi s h and in ve rtebr at es, h o ney bee (Apis m ell{fera), 

### no nt a r ge t in sec ts ( includin g Monarch butte rfl ies) a nd soil in ve rt eb r ates. Add iti o na l s uppl eme nt al studies o n the in sect activity spec trum , a preliminary Tier I V field evaluation , a nd s ummari es of var ious hi g he r tier laborato ry and field s tud ies were also s ubmitted. The se dat a we re s uffi c ient lo a ll ow E PA to determine that th e Cry3 B b I ex pressed in MO 88017 corn presented minimal ri sk to nontarge t o rgani s m s. T he r efore, adverse effects are s imil arl y not expec t ed t o res ult from expos ure to th e Cry3 Bbl ex pre ssed in MO N 8741 I. 

### EPA also co ncl ud e d that ad ve rse eff ect s to f e dera ll y li s t ed threatened and e ndan ge red ("listed ") spec ies were not anticipated, and made a " no effe ct " conc lu s ion for direct and ind irect effect s to I isted s pec ies and th eir d es i gnated critical hab itat s (U .S. EPA 20 I0)
. These assess ments ha ve focused o n li s ted coleopteran spec ies, due t o th e s pe cifi c ity ofC r y3 Bbl to in sec ts of this order. S ince the 20 10 assessment, an additional coleopteran species, Casey's June beet le ( Dina co ma c as ey i ), 

### ha s been li s ted. E PA do es not anticipate any effects to thi s s pecie s be c au se it is I imit ed to s pecific hab it ats, p ri maril y d esert wa s h ar eas with sand y so il s, whi ch wo uld not be provid ed in a r

eas de ve lop ed fo r g row in g co rn. De ve lopment ( primaril y urban o r s uburban ) l eading to destruction of habitat is cited as one of the reaso ns for decl ine of this s pecies and it s cu rre nt restricted ran ge (U .S. FWS 20 11 ), and a r eas where so il is highly di sturbed d o no t pro v id e good habitat (U
. . FW S 2013). Additionally. any adve rse effects t o nontarge t o rga ni s m s a r e expected to be limit ed t o the field in wh ich MON 8741 l is g rown , s in ce pol l e n is not ex pec t e d to move s ig nifi ca ntl y fa r from 

### th e co rn 

### fie ld (U.S. E PA 

### 2 010b) and exp r ess i o n in po ll e n is ve ry low co mp a red to o th e r plant ti ss ues . Sig nifi can t po ll en drift is unlik e ly in a r eas where th e spec ies may occ ur; th e refore. expos ure is not anticip at ed. Therefore. EPA make s a " no effec t'
. de term in a ti o n for direc t a nd indirect eff ec ts fo r Casey's Jun e beetle a nd it s designated critical habitat for C ry3 Bb I expressed in MON 87411. B. Ri s k of No n tar g et Organism s to OvSnn Expre sse d in MO N 87411 C orn 1 . B ird s and Mammal s 

### EPA typically do es not qu antif y expo s ure of no ntarge t ve rteb rates to PIP s, and in s tead reli es o n toxicity tes tin g that is conducted at exposure leve ls r easonab ly ex pected to 

### eq ua l o r, preferabl y, exceed m ax imum expos ure leve ls in the fi e ld based on exp r ess io n s tudi es. Co rn grain is th e p la nt tissu e th a t b irds a nd mammal s a r e mo s t lik e ly to co ns ume, so ir expos ure assump ti o ns a re limited t o g rain o nl y. th e n exposu r e is re lati ve ly simp l e t o unders tand
. However. it is lik e ly that a ll species of birds a nd mammals that inh ab it co rn agroecosys tems are exposed via ot he r so urces. While in c id e nt al dietary ex pos ures t o o th er co rn ti ss ue s may occ ur, exposure thro ug h cons umpti o n of pest inse cts o r othe r inv erteb r ates in se ns itiv e to the toxin s produced b y MON 8741 1 co rn is another route by which bird s a nd m a mmal s m ay be ex po sed lo Ov S n f7
. Whil e OvSnf7 is not expected necessa rily to accumula t e within inv ertebrates , other ti ss ues potential ly co ns um ed by th ese in ve rt eb r a t

es s how exp r ess io n of D vS nf7 a t leve ls severa l o rd e r s of ma g ni tude higher than in g rain. Whi le the leve l or exposu re may no t be s ig nifi ca nt , the ex t ent o f
19

### exposure via this source , or any other possible source , has not been determined. A "worst case " scenario that would reduce uncertainty would be based on the highest concentration shown to be expressed in the P IP plant based on expression studies submitted to EPA. Id eally , exposure in Tier I nontarget organism testing wou ld be derived from these levels. Current toxicity data available to assess risk for birds consists of a 6-week study of broiler chickens fed 57% MON 87411 grain
. If this level of exposure is representative of maximum exposu res in the field, then this study is adequate to address potential effects in birds over the period of time observed. As discussed above , determination of actual exposure may be complicated, and consumption of grain does not represent a " worst case " exposure with complete certainty
. Even if consumed , physiological barriers exist in birds that significantly limit uptake of DvSnt7 , including s alivary and other digestiv e ribonucleas e s (Park et al. 2006, Stevens and Hume 1995) and ac idi c gut environments (Akhtar 2009 , Loretz et al. 2006 , O'Neill et al. 2011 ). Bioinformatic ana lyses with Gallus gal/us 

### and Columba Livia 

### indicated no exact 21 nt matches with the DvSnt7 sequence, providing an additional line of evidence toward expectation of no effects (EPA is cautious , however, in interpreting this informat ion as predictive of effects). Ba s ed on these lines of evidence , adverse effects in birds are unlikely. For a larger scale commerc ial release of MON 874 11 , however , a confi rmatory feeding study with birds should be submitted. Data avai lab le for mammals includes a 28 -d ay study with mice , in which mice were dosed daily at levels up to I 05 m g/ kg/day , and a 90-day study with rats , which were fed MON 87411 corn grain at 33% of the diet for the duration of the study. Both studies indicated no adverse effects
. The dose of 10 5 mg/ kg/day likely exceeds maximum short term exposure levels to mammals that wou ld occur in the field , and 90-day exposu r e to MON 87411 grain provide some insight into longer term exposures at low levels
. As with birds, other lin es of evidence are avai labl e to supplement the data provided , including physiological barriers as discussed above , and bioinformatics anal ysis with mammalian species. Based on these lines of ev idence , the lack of e ffects observed in the avai lable toxicity stud i es , and the limits on the proposed seed increase registration, adverse effects to wild mammal s are not anticipated to result from the proposed registration of OvSnf7 dsRNA in MON 87411. It is noted that E PA has requested a stud y examin in g the s tability of OvSnf7 dsRNA in whole blood to confinn rationale that blood components would break down any dsRNA that may enter the mammalian bloodstream
. S hould these data show that DvSnf7 dsRNA persists in the blood s uch that effects could be considered likely to occur, EPA will require additional effects testing wit h vertebrate species
. 

### 2. Fres hwater Fis h an d Inver teb rates Expos ure in aquatic env ironments may occur as a result of pollen drift to these areas , and a lso potentially through leaf or other post-harvest crop residue movement off of cultivated fields. EP A previously concluded that exposure to Cry protein PlPs in aquatic systems is limited. In the response to the deficiency letter requesting data on DvSnf7 breakdown in plant tissue , Monsanto provided a discussion of the studies supportin g this conclusion and the low likelihood of

20exposu re to senesce nt com plant tissue in aquatic env ironments. This information is suffic ient to sup p o rt this assumption for th e proposed see d increa se regi s tration of MO 1 

### 8741 I. As di sc ussed above, the channel catfi sh study provided some information on the potential dietary toxicity of Dv Snf7 to freshwater fish , but that the usefulne ss of thi s s tudy is limited by the assumptions of ex posure. This s tudy showed that adverse effects a s obse rved arc not expected under s imilar exposure conditions. Since expos ure in aquatic environments is assumed to be minimal based on EPA's current assumpt ions for Cry protein PIP s, then thi s study likel y i s suffic ient to capture potential acute or s ub ch ronic adverse effects of DvSnt7 to fr eshwate r fish at realistic env ironm ental expo s ure level s . Exposure is also expected to be reduced b y the propo se d acreage and tim e limitation s o n the re g i s tration. and expected rapid breakdown of dsR A in so il s. Additionally, E PA notes that expres s ion of DvSnf7 ge nerally declines over th e g r ow in g season, a nd is seve ral orders of magnitude lower in corn s tover and se nescent root. The refo re, level s of DvSnf7 available in corn plants po s t harvest are expected to be lower compared to th e grow in g season. Based on all of this information , adverse effec ts to freshwater fi s h are not expec ted to occur as a result of the propo sed seed increa se registrati o n for D vS nf7 in MON 

### 87411. S imilarl y, s in ce exposure is not expected to be s ignificant in aquatic environments for thi s proposed registration, adverse effects on freshwater invertebrates a re not anticipated. 

### 3. Ma rin e and Es tu a rine Fi s h a nd In ve rteb rates Sign ifi cant exposu re to OvSnf7 i s not expected in marine a nd estuarine env ironments
. Therefore , adverse effects are not anticipated for fi s h or invertebr a te s in these e nvironment s . 4. No ntarge t plant s Exposu re of nonlarget plants to DvSnf7 i s not expected to be s ig nifi can t as di scussed above. Therefore, adverse effects to nontarget plant s are not anticipated. 

### 5. No ntarget In sec t s a nd Other In ve rte brate s Mon santo Company has presented an extensive discu ss ion of the s pecificity of Dv nf7 for it s target pest. Western com rootworm. and it s close r e lati ve. So uthern co m rootworm. Primar y work to demon s trate specificity fo r the target pest i s d esc ribed in Bachman et al. (20 1 3). In thi s study, bioassay s were conduc ted w ith e ith e r DvSnf7 d s RNA or dsRNA developed from an ortho log of th e S11f7 gene in seve ral close r e latives. Thi s s tud y showed that D vSnf7 was on ly active within the Chrysomelidae Famil y of co leopteran s. which provides a strong line of ev idence toward the specific ity of DvS nf7. Exposure to nont a rget in sec t s fo r PIPs is typica ll y based o n express io n levels within plant tissues. Expression levels can differ betw ee n plant tis s ue s and a lso over time. and the plant ti ss ue wi th th e mo s t rele vance to th e in sect diet i s typically used to d etermine dosing le vels or di et concentration in n o ntarget testing. Expression l evels in pollen are often used , si nce many non target insect s will consume pollen , and express ion in leaf may a lso be considered. As de sc ribed above for birds and m a mmal s, the r e i s some uncertainty w ith the actual exposu re in the fi e ld g iven the varying diet s with in arthropod communities. The refore. a conservative appro ac h
21to 

### estimating exposure of nontarget organisms to PIPs would be to utilize the highest expression levels measured among all tissues based on dry weight measurements
. While this may overestimate exposure for certain organisms , it reduces uncertainty around actual exposure in the field
. In the environmental risk assessment submitted by Monsanto Company , NOEC values determined from nontarget testing were compared to maximum expected environmental concentrations (MEECs) to calculate margins of exposure (MOEs). If MO Es are > 1 Ox the MEEC , then risk is assumed to be negligible. Utilizing the highest mean dry weight expression level measured (84.8 x I 0 · 3 

### μg / g whole plant tissue from V3-V4 development stage) , NOEC values of I 000 ng/ g dete1mined in nontarget insect testing would produce a MOE of 11.8, which , if u s ing this approach, would indicate a low likelihood of adverse effects. The MOE for bees 

### would be > 3 ,000, assuming the maximum recorded expression in pollen represents the MEEC for honey bees (maximum dw value measured was 0.292 x 10· 3 

### μg / g). This approach has been found to be acceptable by EPA in previous risk assessments; however, given that the studies are limit dose tests in which only one concentration was tested , there is uncertainty around the biological meaning of the NOEC. Without testing at additional higher concentrations , it is not possible to know how close the NOEC is to levels at which effects that might be observed. Nonetheless , EPA considers level at which toxicity was tested to be sufficiently above expected environmental concentration , and that the studies submitted are reliable tests of potential effect s to nontarget insects at realistic field exposure levels. Monsanto Company supplemented the above data with a discussion of bruTiers to exposure in invertebrate species. 

### It is clear from some studies of dsRNA in invertebrates that some physiological barriers exist that can significantly lim it uptake in insects. These barriers include ribonucleases in saliva (Allen and Walker 2012 , Christiaens et al. 2014) and DNA/RNA nonspecific nucleases in the hemolymph (Garbutt et al. 2013)
. Other evidence of some barriers are demonstrated in studies requiring certain conditions for delivery of dsRNA, such as by microinjection (Whyard et al. 2009) or nanoparticle encapsulation (Sarathi et al. 2008). While these baniers are not fully understood, and information is not yet available to extrapolate between insect taxa , it is apparent that such barriers reduce bioavailability in some insects. Based on the specificity of the intended effect, inve1tebrate studies submitted , and other information provided from the literature, EPA concludes that adverse effects to nontarget insects and other terrestrial invertebrates are not expected to occur as a result of the registration of OvSnt7 in MON 87411 as proposed. Honey bees and other pollinators are included in this conclusion, and the results of testing with both larvae and adult honey bees provide strong ev idence in support. Additionally, Monsanto Company has provided results of bioinformatics analyses with both honey bee and bumble bee , neither of which identified exact 21 nt matches with DvSnt7
. As noted above, these data provide supplemental information to support this concl 

### us ion , but EPA recognizes that they are not necessarily predictive of effects. The potential for off-target and other unintended effects, which were discussed as an uncertainty by the SAP , appears to be unlikely based on the data and other information provided. Understand ing completely all of the potential physiological mechanisms providing barriers to uptake or leading to unintended effects cannot be known without extensive research , which is
22beyond th e scope of E P I\ ·s eco l og ical ri sk assessments. H owever, EPA previously r eq u es te d tes tin g to address potential effects on repro du c ti o n in two additional in sec t s p ec i es . Off-targe t a nd other uninte nd ed effec t s orDvSnf7, if th ey occ ur, a re ex pecte d t o be m o re ev ident in in sec ts, s ince they are more c lose ly r e lated to the tar ge t p cs l. Therefore , thorou g h tes tin g to ensure that a ll port ions of the li fe cycle a re not affected w o uld provide an additional lin e of evidence to co nfirm that unintend e d e ff ec t s of dsRN A would n o t occur in the in sec t spec ies te s ted had the se obse r vations been m ade
. Th ese data s hould be s ubmitted to s upport a Full co mm e rc ia l registration of MO 8741 I.



### 6. Outcro ss in g and Weediness E PA has previou s ly d e te rmin ed that th e re i s no s ig nifi cant ri s k of gene ca pture a nd ex pres s io n of a n y 

### Bt en doto x in by w il d o r wee d y relati ves of co rn in the U
. S
. , it s possessions o r territorie s (see ex te n s ive di sc u ss io n in U.S. EP A 20 I 0). S in ce these co n c lu sio n s are based o n th e n atur e or po llinati o n , s ur viva l of hy brid offs prin g, and weediness in co m and it s relatives, th ese co n c lu s i o n s a l so appl y t o D vS n t7
. 

### 7. Ri s k Asse ss m ent Co nclu s ion s for DvSnf7 Ba se d on the data and ra ti o n a le presented , adverse effec t s to nontar ge t orga ni s m s are not ex p ec te d as a re s ult of th e proposed see d in c rease r eg i stration of Dv S nl7 exp resse d in MON 874 11 com. Add iti onal co nfirmat o ry data as described a b ove wil l be required t o s upport a full commerc ial regi s trati on for M ON 8741 I. EPA ag ree s that the in tended effect of downregulation of the Sn.fl gene i s I ike ly o nl y to be observed in th e targe t insect a nd close r elati ves . The refore, co n c lu s io n s fo r li s ted s p ec i es as de te rmin e d fo r Cry3 Bb I wou ld a l so apply to DvSnf7, s in ce the t a rget in sec t i s a coleopteran s p ec ies. Monsanto Co mp a n y ha s pro v ide d an exte n s ive d isc uss io n t o show th a t the re a li s tic potential for off-targe t e ffect s to other s peci es i s lik e ly to b e ve r y low , and E P A ha s requested con firmatory dat a to s upp o rt thi s conclusion. 

### C. Ri s k of No ntarget Organis m s to C 1 1 1 

### 3Bb l a nd DvSnf7 d s RNA Co mbined a s Expressed in MON 87411 

### Co rn ince exp ress io n level s of Cry3 Bbl do not exceed th ose previously conside r ed in eco log ical risk assess ments, and becau se prcl iminar y data indi cate a lack of sy nergism bet ween th ese P f P s, th e co n c lusions for C r y3Bb 1 and DvSnf7 individu a ll y a l so a ppl y to the combinat ion of these P!P s i n MO N 

### 8741 I for thi s seed in c r ease r egistration. 

### VI. Reference s Akh ta r. S. 2009. O ra l delivery of s iR NA a n d a nti sense o li go nucl eotides. J o urn al of Dru g Ta rget in g 17: 49 1 -495.

23

### Allen , M.L. and W.B. Walker III. 20 12. Saliva of 

### Lygus lineolaris digests double st r anded ribonucleic acids. J ournal of Insect Physiology 58: 391

- 396. Bachman, P. M., R. Bolognesi , W.J. Moar, G.M. Mueller , M.S. Paradise , P. Ramaseshadri , J. Tan , J.P. Uffman, J. Warren, B
.E. Wiggins , and S.L. Levine. 2013. C haracterization of the spectrum of insecticidal act ivity of a double-stranded RNA with targeted activity again st Western corn rootworm 

### (Diabrotica virg!fera virg{fera LeConte). Transgenic Re sear ch 22: 1 207 -1222. Belden, J.B. and M.J. Lydy. 2006. Joint toxicity of chlorpy ri fos and esfenvalerate to fat head minnows and midge larvae. E nvironmental Toxicology and Chemistry 25: 623-629. Christiaens, 

### 0 ., 

### L. Sv. 1 

### evers and G. Smagghe. 20 14
. dsRNA degradation in the pea ap hid 

### (Acyrthosiphon pisum) associated with lack of response in RN Ai feeding and injection assay. Peptides 53: 307-314. Garbutt, J
. S., X. Belles, E.J. Richards , and S
. E
. Reynolds. 2013. Pe rsistence of double-stranded RNA in in sect hemolymph as a potential determiner of RNA interference s ucce ss: Evidence from 

### Manduca sexta and 

### Blatte l/a germanica. Journal of Insect Physiology 59 : 171-178. Kehr , B ., D
. Weese, and B
. Reinert
. 2011. STELLAR : fast and exact local alignments
. BMC Bioinformatics 12 (Suppl 9): S 1 5. Loretz, 

### B. F
. Foger, M
. Werle and A. Bemkop-Schnurch. 2006. Oral gene delivery : S tr ateg ies to improve stability of pDNA towards intestina l digestion 14: 311-319. Macintosh, S
. C., G.M. Kishor, F.J. Periak , P .G. Marrone, T.B. Stone, S.R. Sims, and 

### R.L. Fu chs
. 1990. Potentiation of Bacillus thuringiensis insecticidal activity by serine protease inhibitors. Journal of Ag ricul ture and Food Che mi s try 38 : 1145-1152. O'Ne ill , M.J., L. Bourre , S. Melgar an C.M. O ' D riscoll. 201 1. Intestinal delivery of non-viral gene therapeutics: physio logical barriers and preclinical models
. Drug D iscovery Today 16: 203

- 2 1 8. Park , M.J., Y
. Li, T
. Yu, B.M. Brinkman and D.T. Wong. 2006
. Character izati on of RNA in saliva
. C lini cal C he mi s try 52: 988-994. Sarathi , M., M.C
. S imon, C. Venkatesan and A.S. Hameed. 2008
. Oral administration of bacterially expressed VP28dsRNA to protect 

### Penaeus monodon from white spot sy ndrom e virus
. Marine Biotechnolog y 10: 242 -249. Stevens , C.E. and I.D. Hum e. 1 995. Digestion of ca rb ohydrate, lipids, and protein and the absorption of end products. Page s 152-187 in Comparative Physiology of the Ve r tebrate Digestive System
. Cambridge University Press , Cambridge, United Kingdom.

24

### Tabashnik
.. B.E. 1992. Eva luation of synergism among Bacillus thuringien s is toxins. Applied and Environmenta l Microbiology 58:3343-3346. U
. S. Environmental Prote ct ion Agency (U.S. E PA ). 20 10a. Biopesticides Registration Action Document: Bacillus thuring i ensis 

### Cry3 Bb I 

### Protein and the Genetic Materia l Necessary for 

### It s Production in MON 863 Corn; Bacillus thuringiensis 

### Cr y3Bb I 

### Protein and the Genetic Material Necessary for It s Production in MON 88017 Corn. Biopesticides and Pollution Prevention Division, Office of Pe sticide Pro g ram s . 

### hup :I' ""'" .epa.!!m lpes ti ci<les/biopcsricidc s/rips /crdbb 1-brnd.p<lr. U .S. EPA. 20 I 

### Ob. Bi opesticides Registration Act ion Document: C r y I Ab and Cry IF Bacillus thuringiensis (Bt) 

### Co rn Pl ant- Incorp orated Protectants. Biopesticide s and Pollution Preve ntion Di v is io n , Office of Pesticid e Pro g r a ms. hllp: //v\WW.cp a.!!()\' /()ppbppd I 

### /biopc s ticicl c s/pips /crv I 

### r-cr v I ab-brad.pd I". 

### U.S
. E PA. 2015. Memorandum from J. 

### Facey to 

### J. Kau sc h, Subject: Rev iew or Product Characte ri zation and toxicity data in support for a see d increase Sec. 3 Regi s tration of PlantIncorporated Protectant ( PIP) MO 1 

### 87411 corn, dated August I 0. 20 15. U.S. Fish and Wildlife Service (U .S. FWS)
. 2011. Endangered and Threatened Wildlife and Plants; Dete rmin at ion of End ange r ed S tatu s of Casey's June Beetle, and Designation of C ritical Habitat. United S tates Federal R eg is t er, Vol. 76, No
. 184 , September 22, 2011. http: // wwv.:.!!po.!!ov/ lusys/ pkg/ FR-20 I 1 -09-22/ pd 1/20 I 1-2404 7.pd 

### r. U.S. FWS. 2013. Recove ry Outline for Casey·s Jun e B ee tle (Dinacoma casey i ). March 2013. 

### hllp: l/ ccos.f\vs.!!O\ / docs /rcco\'cn · plan/C.113 Rcco, ·cn Outline F l 1 1 \l..p<lf". 

### Wh yard
.. S. A.D. Singh and S. Wong. 2009. In ges t ed double-stranded R As ca n ac t as specicsspeci fic in secticides. I nsec t Biochemistry and Molecular Biology. 39 : 824-832.

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